CN118562765B - A recombinant Cas12a protein and its use in preparing gene editing and/or epigenetic editing products - Google Patents
A recombinant Cas12a protein and its use in preparing gene editing and/or epigenetic editing products Download PDFInfo
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- CN118562765B CN118562765B CN202410738664.1A CN202410738664A CN118562765B CN 118562765 B CN118562765 B CN 118562765B CN 202410738664 A CN202410738664 A CN 202410738664A CN 118562765 B CN118562765 B CN 118562765B
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Abstract
本发明涉及一种重组Cas12a蛋白及其在制备基因编辑和/或表观遗传编辑的产品中的应用，属于基因编辑技术领域。本发明的重组Cas12a蛋白的氨基酸序列如SEQ ID NO.1所示，是在LbCas12a蛋白基础上发生C175R、F225G、K310L、D350C和V410K的氨基酸突变。本发明的重组Cas12a蛋白具有更广的PAM识别及切割范围和更好的切割活性，能够广泛应用于基因编辑和表观遗传编辑中。
The present invention relates to a recombinant Cas12a protein and its application in the preparation of gene editing and/or epigenetic editing products, belonging to the field of gene editing technology. The amino acid sequence of the recombinant Cas12a protein of the present invention is shown in SEQ ID NO.1, and amino acid mutations of C175R, F225G, K310L, D350C and V410K occur on the basis of LbCas12a protein. The recombinant Cas12a protein of the present invention has a wider PAM recognition and cutting range and better cutting activity, and can be widely used in gene editing and epigenetic editing.
Description
Technical Field
The invention relates to the technical field of gene editing, in particular to a recombinant Cas12a protein and application thereof in preparing a gene editing and/or epigenetic editing product.
Background
CRISPR-Cas nucleases are widely used in gene, epigenetic and base editing of cells and tissues. In addition to the commonly used Cas9 derived from Streptococcuspyogenes (SpCas 9), studies on alternatives to CRISPR nucleases including Cas1, cas8 and Cas9, etc. homologous to Cas12a have found more nucleases with unique and potential advantages.
In Cas12a, asCas a derived from Acidaminococcus sp.bv3l6 strain and LbCas a derived from Lachnospiraceae bacterium ND2006 strain both have ribonuclease activity, and can recognize the target of the T-base-rich Protspacer Adjacent Motif (PAM), and multiple targeting can be achieved by multimerizing crRNA transcripts only with a CRISPRRNA (CRRNA) programming target of 40 nucleotides. Although AsCas a and LbCas a have been used for multiplex gene editing, gene activation and combinatorial library screening, their requirement for PAM recognition of 5' -TTTV (where V is A, C or G) limits the design of relevant crRNA target sites. In addition, the ribonuclease activity of the homologs of Cas12a nucleases, such as FnCas a from FRANCISELLA NOVICIDA U112 strain and MbCas a from Moraxella bovoculi 237 strain, in human cells is not consistent and significant, whereas the LbCas12a variants disclosed in the prior art (such as RVR and RR) are not capable of recognizing 5'-TATV and 5' -TYCV, respectively, but are still incapable of recognizing other PAMs.
Disclosure of Invention
The invention aims to provide a recombinant Cas12a protein and application thereof in preparing gene editing and/or epigenetic editing products, so as to meet the requirements of the prior art on mutant with expanded target recognition capability and recombinant Cas12a protein capable of supporting high target density and high flexibility application.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides a recombinant Cas12a protein, wherein the amino acid sequence of the recombinant Cas12a protein is shown as SEQ ID NO. 1.
The invention also provides a preparation method of the recombinant Cas12a protein, wherein the recombinant Cas12a protein is subjected to amino acid mutation on the basis of LbCas a protein;
The mutation site is the 175 th, 225 th, 310 th, 350 th and 410 th site of LbCas a protein amino acid sequence;
Mutation of amino acid 175 from cysteine to arginine;
Mutation of amino acid 225 from phenylalanine to glycine;
Mutation of amino acid 310 from lysine to leucine;
mutation of amino acid 350 from aspartic acid to cysteine;
Mutation of valine to lysine at amino acid 410;
the amino acid sequence of LbCas a protein is shown as SEQ ID NO. 2.
The invention also provides application of the recombinant Cas12a protein in preparing a gene editing and/or epigenetic editing product.
The invention also provides a nucleic acid molecule for encoding the recombinant Cas12a protein, and the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 3.
The invention also provides a recombinant variant plasmid, which comprises the nucleic acid molecule and an empty vector;
The empty vector is pET-28a (+) vector.
The invention also provides application of the recombinant variant plasmid in preparing a gene editing and/or epigenetic editing product.
The invention also provides a base editor comprising the recombinant Cas12a protein or the recombinant variant plasmid.
The invention also provides application of the base editor in gene editing and/or epigenetic editing.
The invention has the following technical effects and advantages:
The invention is reformed based on LbCas a to obtain the recombinant Cas12a protein with wider range of identifiable PAM, and constructs a base editor with wider range of identifiable PAM, which can aim at PAM which cannot be identified and contacted in the prior art, can realize efficient C > T or A > G editing on the level of cells and embryos for classical PAM and non-classical PAM, and can realize simultaneous multi-site efficient C > T or A > G editing on the level of cells and embryos, thereby enriching the existing base editing tools.
Drawings
FIG. 1 is a recombinant Cas12a protein plasmid map;
FIG. 2 is a pUC57 vector map;
FIG. 3 is a graph showing cleavage activity results of recombinant Cas12a proteins;
fig. 4 is a cleavage range result for a recombinant Cas12a protein.
Detailed Description
The invention provides a recombinant Cas12a protein, wherein the amino acid sequence of the recombinant Cas12a protein is shown as SEQ ID NO. 1.
The invention also provides a preparation method of the recombinant Cas12a protein, wherein the recombinant Cas12a protein is subjected to amino acid mutation on the basis of LbCas a protein;
The mutation site is the 175 th, 225 th, 310 th, 350 th and 410 th site of LbCas a protein amino acid sequence;
Mutation of amino acid 175 from cysteine to arginine (C175R);
Mutation of amino acid 225 from phenylalanine to glycine (F225G);
Mutation of amino acid 310 from lysine to leucine (K310L);
Mutation of amino acid 350 from aspartic acid to cysteine (D350C);
Mutation of amino acid 410 from valine to lysine (V410K);
the amino acid sequence of LbCas a protein is shown as SEQ ID NO. 2.
The invention also provides application of the recombinant Cas12a protein in preparing a gene editing and/or epigenetic editing product.
The invention also provides a nucleic acid molecule for encoding the recombinant Cas12a protein, and the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 3.
In the invention, the nucleotide sequence of the nucleic acid molecule encoding LbCas a protein is shown as SEQ ID NO. 4.
The invention also provides a recombinant variant plasmid, which comprises the nucleic acid molecule and an empty vector;
The empty vector is pET-28a (+) vector.
The invention also provides application of the recombinant variant plasmid in preparing a gene editing and/or epigenetic editing product.
The invention also provides a base editor comprising the recombinant Cas12a protein or the recombinant variant plasmid.
The invention also provides application of the base editor in gene editing and/or epigenetic editing.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the test material, pET-28a (+) vector is purchased from Shenzhenhua large gene technology Co., ltd, competent cells of escherichia coli BL21 (DE 3) are purchased from biological engineering (Shanghai) Co., ltd, ni-NTA agarose purification resin is purchased from GE company, T7 transcription kit is purchased from Saint Biotechnology (Shanghai) Co., ltd, and gene random mutation kit is purchased from Shanghai Biyun biotechnology Co., ltd.
In the test solvent of the present invention:
preparing lysate, namely taking 50mmolTris-HCl and 500mmolNaCl to be constant in 1L of water, and adjusting the pH value to 7.5;
Preparing heavy suspension, namely taking 20mmolTris-HCl to be fixed in 1L of water, and adjusting the pH to 7.0.
EXAMPLE 1 construction of recombinant Cas12a protein plasmid
(1) The primer design is to design a primer LbCas aF/R for synthesizing a LbCas a nucleotide sequence by taking the LbCas a nucleotide sequence extracted from the whole sequence of pMBP-LbCas a obtained from the Addgene website as a template, and to entrust the synthesis of biological engineering (Shanghai) Co., ltd, and to obtain a nucleic acid molecule encoding LbCas a protein by PCR amplification (shown as SEQ ID NO. 4). The nucleotide sequences of the primers are shown in Table 1.
TABLE 1 nucleotide sequence of primer LbCas aF/R
SEQ ID NO.4:
ATGAGCAAGCTGGAGAAGTTTACAAACTGCTACTCCCTGTCTAAGACCCTGAGGTTCAAGGCCATCCCTGTGGGCAAGACCCAGGAGAACATCGACAATAAGCGGCTGCTGGTGGAGGACGAGAAGAGAGCCGAGGATTATAAGGGCGTGAAGAAGCTGCTGGATCGCTACTATCTGTCTTTTATCAACGACGTGCTGCACAGCATCAAGCTGAAGAATCTGAACAATTACATCAGCCTGTTCCGGAAGAAAACCAGAACCGAGAAGGAGAATAAGGAGCTGGAGAACCTGGAGATCAATCTGCGGAAGGAGATCGCCAAGGCCTTCAAGGGCAACGAGGGCTACAAGTCCCTGTTTAAGAAGGATATCATCGAGACAATCCTGCCAGAGTTCCTGGACGATAAGGACGAGATCGCCCTGGTGAACAGCTTCAATGGCTTTACCACAGCCTTCACCGGCTTCTTTGATAACAGAGAGAATATGTTTTCCGAGGAGGCCAAGAGCACATCCATCGCCTTCAGGTGTATCAACGAGAATCTGACCCGCTACATCTCTAATATGGACATCTTCGAGAAGGTGGACGCCATCTTTGATAAGCACGAGGTGCAGGAGATCAAGGAGAAGATCCTGAACAGCGACTATGATGTGGAGGATTTCTTTGAGGGCGAGTTCTTTAACTTTGTGCTGACACAGGAGGGCATCGACGTGTATAACGCCATCATCGGCGGCTTCGTGACCGAGAGCGGCGAGAAGATCAAGGGCCTGAACGAGTACATCAACCTGTATAATCAGAAAACCAAGCAGAAGCTGCCTAAGTTTAAGCCACTGTATAAGCAGGTGCTGAGCGATCGGGAGTCTCTGAGCTTCTACGGCGAGGGCTATACATCCGATGAGGAGGTGCTGGAGGTGTTTAGAAACACCCTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAGCTGGAGAAGCTGTTCAAGAATTTTGACGAGTACTCTAGCGCCGGCATCTTTGTGAAGAACGGCCCCGCCATCAGCACAATCTCCAAGGATATCTTCGGCGAGTGGAACGTGATCCGGGACAAGTGGAATGCCGAGTATGACGATATCCACCTGAAGAAGAAGGCCGTGGTGACCGAGAAGTACGAGGACGATCGGAGAAAGTCCTTCAAGAAGATCGGCTCCTTTTCTCTGGAGCAGCTGCAGGAGTACGCCGACGCCGATCTGTCTGTGGTGGAGAAGCTGAAGGAGATCATCATCCAGAAGGTGGATGAGATCTACAAGGTGTATGGCTCCTCTGAGAAGCTGTTCGACGCCGATTTTGTGCTGGAGAAGAGCCTGAAGAAGAACGACGCCGTGGTGGCCATCATGAAGGACCTGCTGGATTCTGTGAAGAGCTTCGAGAATTACATCAAGGCCTTCTTTGGCGAGGGCAAGGAGACAAACAGGGACGAGTCCTTCTATGGCGATTTTGTGCTGGCCTACGACATCCTGCTGAAGGTGGACCACATCTACGATGCCATCCGCAATTATGTGACCCAGAAGCCCTACTCTAAGGATAAGTTCAAGCTGTATTTTCAGAACCCTCAGTTCATGGGCGGCTGGGACAAGGATAAGGAGACAGACTATCGGGCCACCATCCTGAGATACGGCTCCAAGTACTATCTGGCCATCATGGATAAGAAGTACGCCAAGTGCCTGCAGAAGATCGACAAGGACGATGTGAACGGCAATTACGAGAAGATCAACTATAAGCTGCTGCCCGGCCCTAATAAGATGCTGCCAAAGGTGTTCTTTTCTAAGAAGTGGATGGCCTACTATAACCCCAGCGAGGACATCCAGAAGATCTACAAGAATGGCACATTCAAGAAGGGCGATATGTTTAACCTGAATGACTGTCACAAGCTGATCGACTTCTTTAAGGATAGCATCTCCCGGTATCCAAAGTGGTCCAATGCCTACGATTTCAACTTTTCTGAGACAGAGAAGTATAAGGACATCGCCGGCTTTTACAGAGAGGTGGAGGAGCAGGGCTATAAGGTGAGCTTCGAGTCTGCCAGCAAGAAGGAGGTGGATAAGCTGGTGGAGGAGGGCAAGCTGTATATGTTCCAGATCTATAACAAGGACTTTTCCGATAAGTCTCACGGCACACCCAATCTGCACACCATGTACTTCAAGCTGCTGTTTGACGAGAACAATCACGGACAGATCAGGCTGAGCGGAGGAGCAGAGCTGTTCATGAGGCGCGCCTCCCTGAAGAAGGAGGAGCTGGTGGTGCACCCAGCCAACTCCCCTATCGCCAACAAGAATCCAGATAATCCCAAGAAAACCACAACCCTGTCCTACGACGTGTATAAGGATAAGAGGTTTTCTGAGGACCAGTACGAGCTGCACATCCCAATCGCCATCAATAAGTGCCCCAAGAACATCTTCAAGATCAATACAGAGGTGCGCGTGCTGCTGAAGCACGACGATAACCCCTATGTGATCGGCATCGATAGGGGCGAGCGCAATCTGCTGTATATCGTGGTGGTGGACGGCAAGGGCAACATCGTGGAGCAGTATTCCCTGAACGAGATCATCAACAACTTCAACGGCATCAGGATCAAGACAGATTACCACTCTCTGCTGGACAAGAAGGAGAAGGAGAGGTTCGAGGCCCGCCAGAACTGGACCTCCATCGAGAATATCAAGGAGCTGAAGGCCGGCTATATCTCTCAGGTGGTGCACAAGATCTGCGAGCTGGTGGAGAAGTACGATGCCGTGATCGCCCTGGAGGACCTGAACTCTGGCTTTAAGAATAGCCGCGTGAAGGTGGAGAAGCAGGTGTATCAGAAGTTCGAGAAGATGCTGATCGATAAGCTGAACTACATGGTGGACAAGAAGTCTAATCCTTGTGCAACAGGCGGCGCCCTGAAGGGCTATCAGATCACCAATAAGTTCGAGAGCTTTAAGTCCATGTCTACCCAGAACGGCTTCATCTTTTACATCCCTGCCTGGCTGACATCCAAGATCGATCCATCTACCGGCTTTGTGAACCTGCTGAAAACCAAGTATACCAGCATCGCCGATTCCAAGAAGTTCATCAGCTCCTTTGACAGGATCATGTACGTGCCCGAGGAGGATCTGTTCGAGTTTGCCCTGGACTATAAGAACTTCTCTCGCACAGACGCCGATTACATCAAGAAGTGGAAGCTGTACTCCTACGGCAACCGGATCAGAATCTTCCGGAATCCTAAGAAGAACAACGTGTTCGACTGGGAGGAGGTGTGCCTGACCAGCGCCTATAAGGAGCTGTTCAACAAGTACGGCATCAATTATCAGCAGGGCGATATCAGAGCCCTGCTGTGCGAGCAGTCCGACAAGGCCTTCTACTCTAGCTTTATGGCCCTGATGAGCCTGATGCTGCAGATGCGGAACAGCATCACAGGCCGCACCGACGTGGATTTTCTGATCAGCCCTGTGAAGAACTCCGACGGCATCTTCTACGATAGCCGGAACTATGAGGCCCAGGAGAATGCCATCCTGCCAAAGAACGCCGACGCCAATGGCGCCTATAACATCGCCAGAAAGGTGCTGTGGGCCATCGGCCAGTTCAAGAAGGCCGAGGACGAGAAGCTGGATAAGGTGAAGATCGCCATCTCTAACAAGGAGTGGCTGGAGTACGCCCAGACCAGCGTGAAGCAC
The amino acid sequence of LbCas a protein coded by the polypeptide is shown as SEQ ID NO. 2.
SEQ ID NO.2:
MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH
(2) Plasmid construction by ligating a nucleic acid molecule encoding LbCas a protein into pET-28a (+) vector with reference to the protocol of pET-28a (+) vector to obtain plasmid pET28a-LbCas a, transforming and sequencing, and inserting the combined mutation of C175R, F225G, K310L, D C and V410K into plasmid pET28a-LbCas12a with correct sequencing with reference to the protocol of gene random mutation kit to obtain recombinant Cas12a protein plasmid. Relevant parameters for preparing the recombinant Cas12a protein plasmid are shown in tables 2-3, a map of the recombinant Cas12a protein plasmid is shown in figure 1, and a nucleotide sequence of a nucleic acid molecule encoding the recombinant Cas12a protein is shown in SEQ ID NO. 3.
Table 2 random mutation PCR reaction system
TABLE 3 random mutation PCR reaction procedure for genes
SEQ ID NO.3:
ATGAGCAAACTGGAAAAATTTACCAACTGCTATAGCCTGAGCAAAACCCTGCGCTTTAAAGCGATTCCGGTGGGCAAAACCCAGGAAAACATTGATAACAAACGCCTGCTGGTGGAAGATGAAAAACGCGCGGAAGATTATAAAGGCGTGAAAAAACTGCTGGATCGCTATTATCTGAGCTTTATTAACGATGTGCTGCATAGCATTAAACTGAAAAACCTGAACAACTATATTAGCCTGTTTCGCAAAAAAACCCGCACCGAAAAAGAAAACAAAGAACTGGAAAACCTGGAAATTAACCTGCGCAAAGAAATTGCGAAAGCGTTTAAAGGCAACGAAGGCTATAAAAGCCTGTTTAAAAAAGATATTATTGAAACCATTCTGCCGGAATTTCTGGATGATAAAGATGAAATTGCGCTGGTGAACAGCTTTAACGGCTTTACCACCGCGTTTACCGGCTTTTTTGATAACCGCGAAAACATGTTTAGCGAAGAAGCGAAAAGCACCAGCATTGCGTTTCGCCGCATTAACGAAAACCTGACCCGCTATATTAGCAACATGGATATTTTTGAAAAAGTGGATGCGATTTTTGATAAACATGAAGTGCAGGAAATTAAAGAAAAAATTCTGAACAGCGATTATGATGTGGAAGATTTTTTTGAAGGCGAATTTGGCAACTTTGTGCTGACCCAGGAAGGCATTGATGTGTATAACGCGATTATTGGCGGCTTTGTGACCGAAAGCGGCGAAAAAATTAAAGGCCTGAACGAATATATTAACCTGTATAACCAGAAAACCAAACAGAAACTGCCGAAATTTAAACCGCTGTATAAACAGGTGCTGAGCGATCGCGAAAGCCTGAGCTTTTATGGCGAAGGCTATACCAGCGATGAAGAAGTGCTGGAAGTGTTTCGCAACACCCTGAACCTGAACAGCGAAATTTTTAGCAGCATTAAAAAACTGGAAAAACTGTTTAAAAACTTTGATGAATATAGCAGCGCGGGCATTTTTGTGAAAAACGGCCCGGCGATTAGCACCATTAGCAAATGCATTTTTGGCGAATGGAACGTGATTCGCGATAAATGGAACGCGGAATATGATGATATTCATCTGAAAAAAAAAGCGGTGGTGACCGAAAAATATGAAGATGATCGCCGCAAAAGCTTTAAAAAAATTGGCAGCTTTAGCCTGGAACAGCTGCAGGAATATGCGGATGCGGATCTGAGCAAAGTGGAAAAACTGAAAGAAATTATTATTCAGAAAGTGGATGAAATTTATAAAGTGTATGGCAGCAGCGAAAAACTGTTTGATGCGGATTTTGTGCTGGAAAAAAGCCTGAAAAAAAACGATGCGGTGGTGGCGATTATGAAAGATCTGCTGGATAGCGTGAAAAGCTTTGAAAACTATATTAAAGCGTTTTTTGGCGAAGGCAAAGAAACCAACCGCGATGAAAGCTTTTATGGCGATTTTGTGCTGGCGTATGATATTCTGCTGAAAGTGGATCATATTTATGATGCGATTCGCAACTATGTGACCCAGAAACCGTATAGCAAAGATAAATTTAAACTGTATTTTCAGAACCCGCAGTTTATGGGCGGCTGGGATAAAGATAAAGAAACCGATTATCGCGCGACCATTCTGCGCTATGGCAGCAAATATTATCTGGCGATTATGGATAAAAAATATGCGAAATGCCTGCAGAAAATTGATAAAGATGATGTGAACGGCAACTATGAAAAAATTAACTATAAACTGCTGCCGGGCCCGAACAAAATGCTGCCGAAAGTGTTTTTTAGCAAAAAATGGATGGCGTATTATAACCCGAGCGAAGATATTCAGAAAATTTATAAAAACGGCACCTTTAAAAAAGGCGATATGTTTAACCTGAACGATTGCCATAAACTGATTGATTTTTTTAAAGATAGCATTAGCCGCTATCCGAAATGGAGCAACGCGTATGATTTTAACTTTAGCGAAACCGAAAAATATAAAGATATTGCGGGCTTTTATCGCGAAGTGGAAGAACAGGGCTATAAAGTGAGCTTTGAAAGCGCGAGCAAAAAAGAAGTGGATAAACTGGTGGAAGAAGGCAAACTGTATATGTTTCAGATTTATAACAAAGATTTTAGCGATAAAAGCCATGGCACCCCGAACCTGCATACCATGTATTTTAAACTGCTGTTTGATGAAAACAACCATGGCCAGATTCGCCTGAGCGGCGGCGCGGAACTGTTTATGCGCCGCGCGAGCCTGAAAAAAGAAGAACTGGTGGTGCATCCGGCGAACAGCCCGATTGCGAACAAAAACCCGGATAACCCGAAAAAAACCACCACCCTGAGCTATGATGTGTATAAAGATAAACGCTTTAGCGAAGATCAGTATGAACTGCATATTCCGATTGCGATTAACAAATGCCCGAAAAACATTTTTAAAATTAACACCGAAGTGCGCGTGCTGCTGAAACATGATGATAACCCGTATGTGATTGGCATTGATCGCGGCGAACGCAACCTGCTGTATATTGTGGTGGTGGATGGCAAAGGCAACATTGTGGAACAGTATAGCCTGAACGAAATTATTAACAACTTTAACGGCATTCGCATTAAAACCGATTATCATAGCCTGCTGGATAAAAAAGAAAAAGAACGCTTTGAAGCGCGCCAGAACTGGACCAGCATTGAAAACATTAAAGAACTGAAAGCGGGCTATATTAGCCAGGTGGTGCATAAAATTTGCGAACTGGTGGAAAAATATGATGCGGTGATTGCGCTGGAAGATCTGAACAGCGGCTTTAAAAACAGCCGCGTGAAAGTGGAAAAACAGGTGTATCAGAAATTTGAAAAAATGCTGATTGATAAACTGAACTATATGGTGGATAAAAAAAGCAACCCGTGCGCGACCGGCGGCGCGCTGAAAGGCTATCAGATTACCAACAAATTTGAAAGCTTTAAAAGCATGAGCACCCAGAACGGCTTTATTTTTTATATTCCGGCGTGGCTGACCAGCAAAATTGATCCGAGCACCGGCTTTGTGAACCTGCTGAAAACCAAATATACCAGCATTGCGGATAGCAAAAAATTTATTAGCAGCTTTGATCGCATTATGTATGTGCCGGAAGAAGATCTGTTTGAATTTGCGCTGGATTATAAAAACTTTAGCCGCACCGATGCGGATTATATTAAAAAATGGAAACTGTATAGCTATGGCAACCGCATTCGCATTTTTCGCAACCCGAAAAAAAACAACGTGTTTGATTGGGAAGAAGTGTGCCTGACCAGCGCGTATAAAGAACTGTTTAACAAATATGGCATTAACTATCAGCAGGGCGATATTCGCGCGCTGCTGTGCGAACAGAGCGATAAAGCGTTTTATAGCAGCTTTATGGCGCTGATGAGCCTGATGCTGCAGATGCGCAACAGCATTACCGGCCGCACCGATGTGGATTTTCTGATTAGCCCGGTGAAAAACAGCGATGGCATTTTTTATGATAGCCGCAACTATGAAGCGCAGGAAAACGCGATTCTGCCGAAAAACGCGGATGCGAACGGCGCGTATAACATTGCGCGCAAAGTGCTGTGGGCGATTGGCCAGTTTAAAAAAGCGGAAGATGAAAAACTGGATAAAGTGAAAATTGCGATTAGCAACAAAGAATGGCTGGAATATGCGCAGACCAGCGTGAAACAT
EXAMPLE 2 purification of recombinant Cas12a protein
(1) Prokaryotic expression the recombinant Cas12a protein plasmid obtained in the example 1 is transformed into competent cells of escherichia coli BL21 (DE 3), the competent cells are cultured overnight at 37 ℃ after being coated with LB plates (Kan+), then monoclonal colonies are picked up and inoculated into 100mL of LB culture solution (Kan+), the culture is oscillated at 37 ℃ and 210rpm until OD 600 =0.6 is reached, recombinant Cas12a protein plasmid bacterial solution is obtained, IPTG is added into the recombinant Cas12a protein plasmid bacterial solution to a final concentration of 0.5mmol/L, and the culture is induced for 4 hours under the conditions of 37 ℃ and the rotating speed of 210rpm, so that induced bacterial solution is obtained.
(2) The purification comprises the steps of taking 10mL of induced bacterial liquid, centrifuging for 8min at 8000rpm, taking the precipitate, washing for 2 times by 20mmol/LTris (pH=7.0), carrying out ultrasonic pyrolysis for 4s at 150W power by using 1mL of lysate, standing for 6s, crushing for 1min until the induced bacterial liquid becomes clear, centrifuging for 8min at 8000rpm at 4 ℃, taking supernatant, purifying by using Ni-NTA agarose purification resin, specifically, loading the supernatant onto an equilibrated Ni column at 4 ℃ at a flow rate of 2mL/min through a peristaltic pump, collecting effluent, repeatedly loading for 3h to obtain an Ni column combined with recombinant Cas12a protein, balancing the Ni column combined with the recombinant Cas12a protein again at a flow rate of 2mL/min until the number of an ultraviolet detector is reduced to be stable, and then carrying out gradient elution by using imidazole with concentration of 50mmol/L, 150mmol/L, 250mmol/L and 500mmol/L respectively to obtain the purified recombinant Cas12a protein, wherein the amino acid sequence of the purified recombinant Cas12a protein is shown as SEQ ID NO 1.
SEQ ID NO.1:
MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRRINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFGNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNLNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKCIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSKVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH
Example 3 identification of cleavage Activity of recombinant Cas12a protein
(1) The primer design is to design a primer MPVF3L F/R for synthesizing target DNA by taking a pUC57 plasmid containing a 48048 bp-48509 bp nucleotide fragment of a monkey pox virus F3L gene (NCBIID: AF 380138) as a template, entrusting the synthesis of a biological engineering (Shanghai) stock company, and carrying out PCR amplification for 30min at 37 ℃ to obtain the target DNA. The nucleotide sequences of the primers are shown in Table 4, the PCR amplification system is shown in Table 5, and the map of pUC57 vector is shown in FIG. 2.
TABLE 4 nucleotide sequences of primers and probes
TABLE 5PCR amplification System
| Reagent(s) | Addition of |
| 2×ReactionBuffer | 12.5μL |
| dNTP | 2.3μL |
| 10×BasicE-mix | 2.5μL |
| 10 Mu mol/L primer MPVF3LF | 1.2μL |
| 10 Mu mol/L primer MPVF3LR | 1.2μL |
| 20×CoreReactionMix | 1.25μL |
| Stencil plate | 0.5μL |
| 280Mmol/L magnesium acetate | 1.25μL |
| Water and its preparation method | 2.3μL |
(2) Preparation of crRNA, namely designing a crRNA sequence (shown as SEQ ID NO. 9) according to the characteristics of target DNA, synthesizing the crRNA sequence with a T7 polymerase promoter, and performing in vitro transcription by referring to the use instructions of a T7 transcription kit to obtain the crRNA.
(3) The ssDNA probe is prepared by modifying 5'-FAM group at N-end and 3' -BHQ1 quenching group at C-end, and synthesizing by Shenzhen China big gene technology Co., ltd.
(4) Cleavage Activity identification the anti-cleavage Activity of recombinant Cas12a protein on ssDNA was detected with Simian poxvirus as target site, specifically 125mmol/L, 250mmol/L, 500mmol/L recombinant Cas12a protein independently 15.5. Mu.L, 2.5. Mu.L 0.5. Mu. Mol/LcrRNA, 2. Mu.L NEBuffer2.1, 5. Mu. LssDNA probe and 0.5ng target DNA were added, respectively, and the detection was performed by real-time fluorescence PCR after incubation at 37℃for 2h with no recombinant Cas12a protein (i.e.the concentration of recombinant Cas12a protein was 0 nmol/L) as control. The detection results are shown in FIG. 3.
The results show that the recombinant Cas12a proteins with different concentrations have certain cleavage activity, and the higher the concentration of the recombinant Cas12a protein is, the greater the cleavage activity is.
Example 4 cleavage Range validation of recombinant Cas12a protein
The cleavage range of recombinant Cas12a protein to PAM is verified by taking different crRNAs as targets, specifically taking TTTV, TCCC, TCCA, TATG and TATA as guide crRNA dependent sequences, respectively adding 2.5 mu L of 0.5 mu mol/L recombinant Cas12a protein, 2 mu L of LNEBufer 2.1, 5 mu L of ssDNA probe and 0.5ng of target DNA, and after incubation for 2 hours at 37 ℃, detecting by adopting real-time fluorescence PCR. The nucleotide sequence of the guide crRNA-dependent sequence is shown in Table 6, and the detection results are shown in FIG. 4.
TABLE 6 nucleotide sequence of guide crRNA dependent sequences
| Name of the name | Sequence (5 '. About.3') | SEQIDNO. |
| TTTV | TTTATGCCTGTGTAGACATTGACGG | 11 |
| TCCC | TCCCATGCCTGTGTAGACATTGACGG | 12 |
| TCCA | TCCAATGCCTGTGTAGACATTGACGG | 13 |
| TATG | TATGATGCCTGTGTAGACATTGACGG | 14 |
| TATA | TATAATGCCTGTGTAGACATTGACGG | 15 |
The results indicate that the recombinant Cas12a protein has certain cleavage activity on TTTV, TCCC, TCCA, TATG and TATA, and the cleavage activity of the recombinant Cas12a protein on the guide crRNA-dependent sequence is ordered as TATG > TATA > TCCA > TTTV > TCCC.
From the above examples, the present invention provides a recombinant Cas12a protein and its use in the preparation of gene editing and/or epigenetic editing products. The recombinant Cas12a protein has wider PAM recognition and cutting range and better cutting activity, and can be widely applied to gene editing and epigenetic editing.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. The recombinant Cas12a protein is characterized in that the amino acid sequence of the recombinant Cas12a protein is shown as SEQ ID NO. 1.
2. The method of preparing a recombinant Cas12a protein of claim 1, wherein the recombinant Cas12a protein is mutated in amino acids based on LbCas a protein;
The mutation site is the 175 th, 225 th, 310 th, 350 th and 410 th site of LbCas a protein amino acid sequence;
Mutation of amino acid 175 from cysteine to arginine;
Mutation of amino acid 225 from phenylalanine to glycine;
Mutation of amino acid 310 from lysine to leucine;
mutation of amino acid 350 from aspartic acid to cysteine;
Mutation of valine to lysine at amino acid 410;
the amino acid sequence of LbCas a protein is shown as SEQ ID NO. 2.
3. Use of the recombinant Cas12a protein of claim 1 for the preparation of a gene-edited and/or epigenetic edited product.
4. A nucleic acid molecule encoding the recombinant Cas12a protein of claim 1, wherein the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID No. 3.
5. A recombinant variant plasmid comprising the nucleic acid molecule of claim 4 and an empty vector;
The empty vector is pET-28a (+) vector.
6. Use of the recombinant variant plasmid according to claim 5 for the preparation of a gene-edited and/or epigenetic edited product.
7. A base editor comprising the recombinant Cas12a protein of claim 1 or the recombinant variant plasmid of claim 5.
8. Use of the base editor of claim 7 for gene editing and/or epigenetic editing.
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